Existing protein analysis technology is largely based upon two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), which has undeniably assumed a major role and is central to much of what is now described as “proteomics.” Typically, proteins are separated by charge in a first dimension, based on isoelectric focusing in a pH gradient medium, and by size in a second dimension, based on molecular weight in a polyacrylamide gel containing sodium dodecyl sulfate (SDS). When proteins are radiolabeled, or stained, their positions in the gel are detected by autoradiography, or densitometry, respectively.
Despite the selectivity of 2-D PAGE, existing techniques are a collection of manually intensive procedures and time-consuming tasks prone to irreproducibility and poor quantitative accuracy. Thus, automated, high resolution, rapid, reproducible, and ultrasensitive 2-D separation techniques would be advantageous for large-scale analysis of proteins.
Microfluidic platforms offer fast, accurate, and low cost electrokinetic systems for high-throughput 2-D PAGE. One drawback of existing systems is a lack of methodology to detect protein separations in microchannels. Performance of the isoelectric focusing and the size based separation can be monitored by detecting the proteins in microchannels. A robust detection system of proteins in microchannels, is not only important for identification of proteins, but also important for quantification of proteins, with accuracy and resolution.
Another drawback of the application of existing microfluidic techniques to 2-D PAGE devices is a lack of methods to introduce different separation media into different dimensions in the same unit. Performing both charge and size based separations in one miniaturized 2-D PAGE device is desirable for high-throughput purpose.
Another drawback of the application of existing microfluidic techniques to 2-D PAGE devices is a lack of methods to transfer proteins simultaneously from first to second dimensions without significant loss in resolution. In existing methods, protein analytes are continuously sampled in the first dimension and transferred to the second dimension. To date, sufficient resolution has not been achieved using existing methods.
A problem with microfluidic devices for 2-D DNA gel electrophoresis is the lack of convenient, effective methodology to transfer DNA molecules from a first dimension to a second dimension after separation of molecules in the first dimension. Microfluidic devices for 2-D DNA gel electrophoresis also suffer from the lack of a convenient method or device for high throughput and high resolution second dimension separation. Current approaches using DGGE or other currently available gel based methods for this sequence-dependent separation in microfluidic devices have limitations in handling for high throughput purposes.
These and other drawbacks exist.